Role of Chemokines During the Different Phases of Rheumatoid Arthritis

A great deal of the data available thus far regarding the pathogenic mechanisms in arthritis has been derived from animal models. For instance, the identification of TNF-a as a therapeutic target, one of the effective immunotherapeutic agents in RA so far, was established in studies conducted in animal models (5,9,10). Collagen type II (CII)-induced arthritis (CIA) in the arthritis-susceptible DBA/1j mouse strain is one of the most commonly used immunization-based models in which many of the pathologic features of human RA are recapitulated (11-16). The role of chemokines during the four phases described above can be clearly appreciated in the CIA model.

10.2.1. Induction Phase

During this phase, chemokines and their receptors could play a very important role by promoting cellular encounters in lymphoid structures. Although a growing body of evidence supports the notion that DCs play a major role in the initiation and perpetuation of RA by presenting presumed arthritogenic antigens to autoreactive T cells (17-20), the precise mechanisms by which DCs affect disease pathogenesis remain to be defined. DCs may capture the antigen (Ag) in the periphery (in the case of CIA, the Ag is injected intradermally) and migrate toward the draining lymph node (DLN). This migration is guided by chemokine gradients. In particular, upregulation of CCR7 in DCs, a phenomenon that occurs as part of their "maturation" process, confers responsiveness to high levels of the CCR7 ligand CCL21 that is upregulated in the lymphatic channels and in lymph nodes (LNs) (21). Also, another CCR7 ligand, CCL19, is abundantly expressed in the T-cell zone of the LN, and this chemokine attracts Ag-transporting DCs to the T-cell zone (21). In the T-cell zone, DCs encounter T cells responsive to CII epitopes.

One of the more important features of CIA is the production of anti-CII antibodies, which play an important role during the effector phase of the disease. After activation by DCs, CII-specific T cells encounter B cells that are present in the B-cell follicles of LNs. This interaction may depend on the upregulation of the chemokine receptor CXCR5, among others, in some activated CII-specific T cells, which leads to their migration toward the high concentration of the CXCR5 ligand and CXCL13 in the B-cell follicles (21,22). Ag-specific B- and T-cell interaction may lead to the formation of germinal centers where B cells may become very efficient in producing Abs and begin their differentiation into plasma cells. Plasma cells commonly reside in the bone marrow (BM). Therefore, it is likely that some of the CII-specific B cells may upregulate CXCR4 and migrate toward the high levels of the CXCR4 ligand CXCL12 that are present in the BM microenvironment (23).

10.2.2. From Effector Phase to Maintenance

At the conclusion of the induction phase in CIA, CII-specific T cells and plasma cells producing high levels of anti-CII antibodies would initiate the early effector phase of the disease. Recently, Wipke et al. (24) have proposed a four-step model to describe how certain autoantibodies promote the effector phase of arthritis (Fig. 1; see color plate): In the first stage, Abs in circulation may form immune complexes (ICs) with their cognate Ags. These ICs would bind to FcyRIII on neutrophils triggering the release of vasoactive mediators such as TNF-a, resulting in increase in vascular permeability at the level of the joints. Some IC-bound neutrophils would enter the perivascular space where they encounter other cells that may further amplify the disease process in the joints (e.g., mast cell). In the first stage, it is likely that neutrophil-attracting chemokines play a major role. In the second stage, additional Abs would bind to their target leading to activation of innate effector mechanisms and inflammation. For instance, Abs bound to the cartilage surface activate the alternative pathway of complement, producing the anaphylotoxin C5a; C5a then activates

Table 1

Chemokine or Chemokine-Receptor Antagonists in Clinical Trials

Target

Name

Company

Status

Disease/comments

Ref.

CCL2

ABN912 (monoclonal antibody)

NOVARTIS

Phase I/II

Asthma. RA. In RA patients, there was increase dsynovial macrophages and CCL2 circulating levels.

95, 109

CCL11

Bertilimumab

Cambridge AT

Phase II

Conjunctivitis, allergic rhinitis

110-113

(Eotaxin)

(CAT-213) Monoclonal IgG4

CXCL8

Monoclonal Ab

XenoMouse

Phase II

RA. No clinical benefits

114, 115

(IL-8)

anti-IL8

Technology

BX-471 (ZK-811752)

Berlex Biosciences/

Phase II

MS, psoriasis, eczema

111-113,

(nonpeptide)

Shering AG

Phase I

Alzheimer disease

116, 117

MLN-3897

Millenium Pharmaceuticals/ Aventis

Phase II

RA, MS, psoriasis

111, 112, 118

Pharmaceuticals

Phase I

RA

111, 112, 119

Unknown

Pfizer

Phase I

RA. Reduction in joint

120

CCR1

inflammation, no. of macrophages and CCRI cells

in synovium, decreases in overall cellularity, CD4+ or CD8+ T cells. However, no clinical benefits.

in synovium, decreases in overall cellularity, CD4+ or CD8+ T cells. However, no clinical benefits.

CP-481,715 Pfizer

C-4462

CCR2

CCR3

g CCR5

CXCR1/2 CXCR3

CXCR4

Banyu (Merck) MLN-1202

(antibody) INCB3284

GW-766994

DPC-168 UK-427857

(MARAVIROC) ONO-4128

Sch-351125 / Sch-417690 SB-332235 T0906487

AMD-3100

AMD-070 CTCE-0214

Phase II Millenium

Phannaceuticals Incyte Pharmaceuticals/

Pfizer GlaxoSmithKline

Bristol-Myers Squibb Pfizer

Pharmaceutical/

GlaxoSmithKline Schering-Plough

GlaxoSmithKline Tularik-Amgen and Chemocentryx AnorMED

AnorMED Chemokine Therapeutics

RA. Inhibits 90% of monocyte chemotactic activity present in 11 of 15 RA synovial fluid samples.

RA 122

Phase II RA

Phase IIa RA, obese insulin-resistant. It also presents mild anti-CCR5 effect.

Phase II Asthma, allergic rhinitis

Phase I Asthma

Phase II HIV infection

Phase II HIV infection

Phase I HIV infection

Phase I COPD, RA, psoriasis

Phase Ha Psoriasis. Was suspended because no effect was found. Phase II Stem cell transplantation Phase I Repair cardiac tissue after myocardial infarction Phase I HIV infection Phase II Stem cell transplantation

124 111-113,

125 111, 112 111-113,

126-128 111, 112

111, 112

111, 112 111, 129

111, 112 111, 112

111, 112 111, 112

COPD, chronic obstructive pulmonary disease; MS, multiple sclerosis; RA, rheumatoid arthritis.

multiple cell types (neutrophils, mast cells, macrophages, and endothelial cells) to produce proinflammatory molecules (i.e., TNF-a, IL-1, and chemokines), thereby amplifying the inflammatory cascade (third stage). In the last stage, after prolonged inflammation, chronic changes in the joint occur including activation of osteoclasts, resulting in bone erosion, and formation of lymphoid tissue aggregates (see below). Locally, monocytes recruited from the circulation will differentiate into inflammatory macrophages. Also as part of the effector phase, CII-specific T cells may produce cytokines that promote the differentiation of bone-reabsorbing cells known as osteoclasts, for example receptor activator of nuclear factor kappa B ligand (RANKL) and TNF-a (25,26). This chronic stage would in many cases imply the progression toward the maintenance phase of the disease (Fig. 1; see color plate).

Probably among all the phases of the disease, the effector and maintenance phases of RA are the ones for which we have the most information regarding the role of chemokines. Several studies have demonstrated that in the actively inflamed synovial membrane of patients with RA, their inflammatory synovial fluid, peripheral blood mononuclear cells (PBMCs), and serum/plasma samples, there is broad upregulation of the levels of several chemokines and their receptors (Table 2). In few studies, the specificity for RA in the upregulation of chemokines has been demonstrated by comparing the RA samples with those obtained from other processes affecting the joints such as osteoarthritis or infectious forms of arthritis. For instance, in synovial tissue derived from patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis, abundant expression of CCR1, CXCR4, and CCR5 has been reported. Notably, tissue levels of CCL5 and CCL15 are higher in RA than osteoarthritis or reactive arthritis (27) (Table 3).

10.2.3. Formation of New Lymphoid Aggregates in the Inflamed Synovial Tissue

One of the more interesting processes that chemokines may drive during the effector and maintenance phases of RA is the formation of lymphoid aggregates in the synovium, known as lymphoid neogenesis. This involves the formation of organized B-cell follicles containing germinal centers and T-cell areas (28). These newly formed lymphoid organs are embedded in the tissue of the joints and lack afferent lymph vessels. A key characteristic of the lymphoid structures in the joints is that in contrast with other structures such LNs, persistent antigenic stimulation is required to induce and maintain them (28). Interestingly, formation of lymphoid aggregates has also been described in the joints of mice with CIA (29).

The molecular mechanisms that underlie the formation and maintenance of lymphoid structures such as LNs and those in chronically inflamed synovium

Chemokines and Chemokine Receptors Implicated in Rheumatoid Arthritis: Human Studies

Molecule

Location

Cell

Comments

Detection

Ref.

CCL2

Serum

EC

SF

SC

Synovium

ELS, CD68

TB

OB, osteocytes, SC,

EC, MNC

CCL3

Synovium

EC

SF

Mo, PMN

PBMCs

MDDC

TB

OB, MNC

FDL

|CCL4

Synovium

o

TB

MNC

SCCL5

Serum

ELS

u

Synovium

SC (nonendothelium)

TB

OB, MNC

CCL17

SF

PBMCs

MDDC

CCL18

SF

Synovium

PBMCs

MDDC

CCL19

Synovium

CCR5 ligand

PBMCs

MDDC

CCR2 ligand

CCR5 ligand

IF, ELISA, IHC

CCR5 ligand Main CCR5 ligand

CCR4 ligand. Th2 chemokine

Expressed in DC. Can act as antagonist of CCL11 and CCL13 for binding to CCR3.

CCR7 ligand

PBMCs

MDDC

Real-time PCR (mRNA), IHC, ELISA

IHC, ELISA

Real-time PCR (mRNA), ELISA, IHC, IF Real-time PCR,

ELISA ELISA, real-time PCR (mRNA), IHC

139, 145

Molecule Location

Cell

CCL20 Synovium, SF

CCL21

CCL22

Synovium, subcondral BM, serum, SF SF

FLS CD3, CD20, EC

CCL28 Synovium

1CXCL1

ICXCL5 u CXCL6 CXCL8

Synovium

TB Synovium SF

Serum, urine, SF, synovium FDL PBMCs TB

CXCL9 Synovium

MDDC

OB, osteocytes, EC,

Comments

Detection

Ref.

Controls migration of immature DC through CCR6

Involved in the organization of secondary LN (ectopic LN in synovium)

Detection restricted to juvenile RA samples

Ligand for CCR10 (present on B cells)

Neutrophil migration, acute inflammation, innate immunity

Real-time PCR, RT-PCR, ISH, ELISA, IHC IHC, IF, RT-PCR, ISH (mRNA), ELISA

ELISA

IHC Real-time PCR

(mRNA), IHC IHC, IF, ELISA ELISA, IHC Real-time PCR (mRNA), ELISA, IHC, ISH

32, 146-149

144 154

131, 155

156 132

155-158

CXCR3 ligand

RT-PCR, FACS, IHC, ELISA, DNA microarray

EpC,STC pDC

CXCL11 Synovium SF

CXCL12 Synovium SF TB

3CXCL13

Cartilage Synovium, subcondral BM Synovium

CXCL16 Synovium, SF

SC (nonendothelium),

STC pDC

Chondrocytes CD3, CD20, FDC

XCL1

CX,CL1

Synovium SF

Synovium SF

MCCRL2

CCR3

PBMCs, SF SF, synovium

PBMCs SF, synovium

CD 14

Stromal derived factor. Involved in development of CNS and vasculature; B-cell lymphopoiesis. Involved in the organization of secondary LN (ectopic LN in synovium)

IHC, IF, real-time PCR (mRNA), ELISA, RT-PCR, DNA microarray Real-time PCR (mRNA), IF, ELISA, RT-PCR Real-time PCR (mRNA), RT-PCR, Northern, ISH, IHC, IF, ELISA RT-PCR, IHC, ISH (mRNA)

Membrane bound form can act as adhesion molecule Secreted by T cells and involved in T-cell traffic. Thl chemokine.

IHC, RT-PCR 176-178

FACS, ELISA

Orphan receptor Marker for Th2 cells

IHC IHC

FACS, IHC

179-181 182

77, 180

Molecule

Location

Cell

Comments

Detection

Ref.

CCR4 SF, PBMCs

CCR5

CCR6

g CCR7

CXCR1 CXCR2 CXCR3

PBMCs, SF, synovium

Serum,

Synovium.dral BM.V KJ,n?

synovium, STC, SF PBMCs, SF, synovium

Synovium Synovium Synovium, SF

CD4+ (CD28), CD4+ (CD45RO), CD4+ (CD45RA); FLS, CD3, CD4, CD8, CD 14 CD4 (CD45RO), DC

Marker for Th2 cells

T-cell Chemotaxis (effector memory)

Homing to LN and synovium

(central-effector memory) Effector memory Central memory

Homing to inflamed tissue

FACS, IHC

Real-time PCR, FACS, ISH, IHC

RT-PCR, WB, IHC RT-PCR, WB DNA microarray, RT-PCR, FACS, IHC, IF, WB

35, 47, 77, 130, 137, 138, 144, 150, 179-181, 184 32, 146-148

159 159

CXCR4

OS On

DARC

PBMCs, SF, synovium

CXCR6 Synovium,

PBMCs, SF CXjCRl PBMCs, synovium, SF

Synovium Synovium

CD4+ (CD28), CD4+ (CD45RO), CD8+ (CD45RO), EC, pDC, MLS, FLS

(CD45RA") CD3, CD4, CD4 (CD28), CD8, CD 14, MLS, FLS, DC, EC

MNC EC (venules only)

Homing to LN and synovium

(central-effector memory) Effector memory Central memory

Present on activated memory

Thl cells Interaction between ligand and receptor can mediate strong cell adhesion, and induce T-cell activation, cytotoxicity, and proliferation

Possible role in transmigration of cells from blood to inflamed tissue

FACS

RT-PCR, ISH

Northern, IHC

35, 108, 130, 150, 162, 164-166, 179, 181, 184 130, 173

176-178, 180

138, 175 185, 186

DC, dendritic cell; EC, endothelial cell; EpC, epithelial cell; FDC, follicular dendritic cell; FDL, foot draining lymph; FLS, fibroblast-like synoviocyte; MC, mast cell; MDDC, monocyte-derived dendritic cell; MLS, macrophage-like synoviocyte; MNC, mononuclear cell; Mo, monocyte; M0, macrophage; OB, osteoblast; pDC, plasmacytoid dendritic cell; PMNs, polymorphonuclear neutrophils; SC. stromal cell; STC, synovium tissue cell; TB, trabecular bone.

Key Chemokines and Chemokine Receptors Relevant in Rheumatoid Arthritis Compared with Other Arthritides:

Human Studies

Molecule

Sample

Detection

Comment

Ref.

CCL2

Serum

ELISA

CCL3

CCL5

CCL8 CCL17

Multiplex IHC

Synovium, SF ELISA IHC

Serum Multiplex

Synovium, SF ELISA

Serum SF

Synovium SF

Synovium

ELISA

ELISA ISH (mRNA)

ELISA

Real-time PCR

Increased levels compared with OA samples.

Highest levels present in patients with follicular synovitis compared with those with diffuse synovitis.

Similar levels to inactive SLE but reduced when compared with active SLE.

Levels comparable with RP.

Comparable levels found in early arthritis (<ly), and late arthritis (>5y).

Higher levels compared with OA, gout, and traumatic joint injury.

Higher levels found in early arthritis (<ly), in lining and sublining layer of synovium compared with late arthritis (>5 y).

Levels comparable with RP.

Higher levels compared with OA and traumatic joint injury.

Increased levels compared with OA samples.

Highest levels present in patients with follicular synovitis compared with those with diffuse synovitis.

Higher levels compared with OA and gout.

Increased expression in the cellular infiltrate compared with OA but not PA.

Higher levels compared with OA or other inflammatory arthritides.

Elevated levels compared with OA or normal controls.

38, 187

CCL18 Serum, drain fluid, SF

Serum

Multiplex

CCL19 CCL20 CXCL1

CXCL6

gCXCL8

Synovium SE, SF Synovium

Serum

Real-time PCR ELISA ISH (mRNA)

ELISA

ELISA

SPCIA ELISA

CXCL9

Synovium SF, synovium

Real-time PCR

ELISA DNA microarray, RT-PCR, ELISA, ISH (mRNA)

Higher levels present in SF of RA patients compared 139, 145, 187, 191 with serum of normal donors and drain fluids from lymph node resection of mammary carcinoma.

Higher levels compared with gout and OA.

Higher levels compared with normal controls but lower when compared with RP.

Elevated levels compared with OA or normal controls. 139

Elevated levels compared with OA. 147-149

Increased expression in the synovial lining layer 190

compared with OA but not PA.

Higher levels compared with OA or other inflammatory 132 arthritides.

Increased levels compared with OA samples. Highest 38, 133, 139, 156, levels present in patients with follicular synovitis 189, 191-196

compared with those with diffuse synovitis.

Increased levels compared with AOSD and normal controls.

Decreased levels in ERA JA compared with RA.

Similar levels between polyarticular JA and RA.

Higher levels found in polyarticular JA compared with ERA JA.

Higher levels compared with OA and traumatic joint injury.

Comparable or higher levels with gout.

Elevated levels compared with OA or normal controls.

Elevated levels compared with tissue obtained from amputation for diabetes or other etiologies.

Higher levels compared with OA and traumatic joint 38, 159, 160, 190 injury.

Increased expression compared with OA but not PA.

Molecule

Sample

Detection

Comment

Ref.

CXCL10

SF, synovium,

DNA microarray,

Higher levels compared with OA and traumatic joint

38, 159, 160, 162,

serum

RT-PCR, ELISA

injury but not PA.

163

CXCL11

SF, synovium

ELISA, RT-PCR

Higher levels compared with OA but not PA.

160, 162

CXCL12

Serum

ELISA

Elevated levels in RA and OA, significant reduction after synovectomy.

166, 197

XCL1

Synovium

RT-PCR (mRNA), ISH (mRNA), IHC

Higher levels compared with OA. Expression restricted to lymphocytic infiltrate in RA. Immunostaining shows strong staining in RA compared with OA and reactive arthritis but not PA.

175

CXjXLl

SF

ELISA

Higher levels compared with OA, JRA, PA, polyarthritis, and gout.

178

CCR4

PBMCs

FACS

Increased levels of CD4 CCR4 found in active RA, AS, and untreated SLE compared with controls.

183

CCR5

SF

FACS

Increased ratio of CCR5/CCR3 of T cells (CD8) in RA compared with other arthritides.

198

CXCR1

Synovium

DNA microarray, RT-PCR, WB

Higher levels compared with OA.

159

CXCR2

Synovium

DNA microarray, RT-PCR

Higher levels compared with OA.

159

CXCR3

Synovium

DNA microarray,

Higher levels compared with OA.

RT-PCR, WB

AOSD, adult onset Still disease; AS, ankylosing spondylitis; CA, crystal-induced arthritis; ERA, enthesitis-related arthritis; JA, juvenile arthritis; PA, psoriatic arthritis; RA, rheumatoid arthritis; SE, synovium explants; SLE, systemic lupus erythematosus; SPCIA, solid phase 2 site chemilu-minescent immunometric assay; RP, relapsing polychondritis.

are thought to be very similar (28) and mainly driven by the cytokine lymphotoxin and chemokines such as CCL19, CCL21, CXCL12, and CXCL13, which, as we reviewed above, regulate DC and lymphocyte homing and compartmentalization (28). Chronic antigen stimulation leads to (i) persistent activation of innate and adaptive immune cells in the inflamed tissue;

(ii) increased expression of lymphotoxin by activated B and T cells; and

(iii) increased lymphoid chemokine expression by resident stromal cells, infiltrating macrophages, DCs, and other parenchymal cells. Synthesis of lymphoid chemokines might be induced by lymphotoxin itself. Recruitment of B cells, T cells, and DCs to the newly formed lymphoid structures is thought to be facilitated by acquisition of a high endothelial venule (HEV)-like phenotype by the activated endothelial cells in the joint. CCL19 and CCL21 produced by stromal cells (probably fibroblasts or fibroblast precursors) would favor the formation of T-cell areas (28,30). Under the influence of lymphotoxin, stromal cells would acquire the phenotypic and functional properties of a follicular dendritic cell (FDC), including CXCL13 production, which, as mentioned earlier, promotes germinal-center organization (28,31). Interestingly, a similar role for DC in disease initiation in the LN and perpetuation in the newly formed lymphoid tissue in the synovium has been postulated and would be directed by a similar array of chemokines and chemokine receptors (i.e., CCL21, CCL19, and CCR7) (32).

10.2.4. Chemokines in the Migration of Arthritogenic T Cells

Naive T cells migrate back and forth from the blood into the LN, and effector T cells migrate mainly from the blood into the inflamed tissues (i.e., the inflamed synovium) (33,34). An increasing body of evidence suggests that after their activation by APCs in the LN, some T cells will become effector cells and migrate to the sites of inflammation, in the case of RA to the joints, whereas other T cells would become memory T cells. Some of these memory T cells have been termed central memory T cells, because they maintain the ability to migrate into the LN and expand rapidly after exposure to their cognate Ag (33,34). T cells accumulating in RA-inflamed synovial tissue are likely to represent both short-lived effector memory cells and long-lived central memory T cells. The latter cell type finds a niche in LNs. By maintaining CCR7 expression, these end-differentiated T cells can still home to lymphoid organs, enhance their survival, support clonal expansion, and perpetuate autoreactivity (35).

With regard to the specific accumulation of effector T cells in the inflamed RA joints, several papers have documented that T cells in the joints of individuals with RA preferentially express CCR5 (36-38). Furthermore, RA (39) and CIA are thought to be a T helper 1 (Th1)-driven diseases (12,40), and expression of CCR5 is considered to be the hallmark of Th1 differentiation, both in human and mice (41,42). With this data in mind, it was surprising that absence of CCR5 in the DBA/1j background did not modify the course nor the incidence of CIA (43), arguing against the current notion that CCR5 is a central player in RA pathogenesis. It is noteworthy, however, that in the context of a milder disease phenotype as can be seen in the B6x129 mice immunized with CII and complete Fruend's adjuvant (CFA), CCR5 gene inactivation has been shown to have a minor positive effect: whereas the incidence of arthritis was 24.5% in the wild type, the incidence in CCR5 knockout (KO) mice was 9.3% (44). However, arthritis scores and days of onset and duration were not statistically different between the two groups (44).

One explanation for the phenotype observed in the CCR5 null mice may be because CCR5 is not the only chemokine receptor whose expression is elevated in T cells present in the inflamed RA joints. For example, invariably both CCR5 and CXCR3 (and their ligands) are coelevated in RA (36-38). Indeed, although pharmacologic antagonism of CCR5 leads to partial protection against the disease in the CIA model, it is important to note that the antagonist used in these studies blocks not only CCR5 but CXCR3 as well (45), making it difficult to ascribe the protective effect solely to the antagonism of CCR5. Indeed, specific blockade of CCR5 with a monoclonal antibody (MC-68), either in early or late disease phase, did not significantly influence the arthritis score (46).

Finally, Santiago et al. (47) demonstrated that lymphocyte chemoattraction induced by rheumatoid arthritis synovial fluid is independent of CCR5. They showed that T cells from individuals with CCR5 delta 32 mutation (a 32-base-pair frameshift deletion resulting in absent CCR5 expression) migrate normally to chemokines present in the synovial fluids of RA patients (47).

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